1. Field of the Invention
The present invention relates to a process for separating and purifying two sweetening substances of aspartame (APM) and N-[N-(3,3-dimethylbutyl)-L-(xcex1-aspartyl]-L-phenylalanine methyl ester, which hereinafter may be referred to as xe2x80x9cN-(3,3-dimethylbutyl)-APMxe2x80x9d or xe2x80x9cAPM derivativexe2x80x9d, by separating the two substances from each other.
2. Description of the Background
In recent years, eating habits have improved as awareness of obesity caused by excessive sugar intake and diseases accompanied by fatness has increased. Accordingly, the development of a low-calory sweetening agent (sweetener) that replaces sugar has been in demand. As a sweetening agent that is widely used at present, there is aspartame (APM) which is excellent in safety and quality of sweetness. However, the stability thereof remains problematic.
Under these circumstances, French Patent No. 2697844 describes the evaluation of derivatives in which an alkyl group is introduced on an amino group of aspartic acid constituting the APM in one approach to improve slightly the stability and to improve the sweetening potency. N-(3,3-dimethylbutyl)-APM is noted as markedly improving in the sweetening potency. For the production of N-(3,3-dimethylbutyl)-APM, a process for alkylating APM reductively under the coexistence of 3,3-dimethylbutylaldehyde with sodium cyanoborohydride in methanol (refer to the abovementioned FR 2697844 specification), and a process for alkylating APM reductively under the coexistence of 3,3-dimethylbutylaldehyde with platinum carbon as the catalyst in a mixed solvent of water and methanol at a pH value in a range of 4.5 to 5 (WO95/30689) are known. However, when a reaction is carried out according to the processes described in these patent specifications, 3,3-dimethylbutylaldehyde and APM are either both unreacted, or N,N-di(3,3-dimethylbutyl)-APM, produced as a by-product, wherein two alkyl groups have been introduced thereto, are mixed in the reaction solution (mixture) or the crude crystals of the product, to no small extent.
Among the compounds described, 3,3-dimethylbutylaldehyde is removed by drying under reduced pressure or washup with comparative ease, because it has a low boiling point or is soluble in a poor solvent for the N-(3,3-dimethylbutyl)-APM, such as hexane. On the other hand, it is difficult to remove APM and N,N-di(3,3-dimethylbutyl)-APM by crystallization from the residue from which the catalyst and the like have been removed as described in the above-mentioned patent specifications. Hence, the yield thereof is much reduced in order to obtain N-(3,3-dimethylbutyl)-APM having a satisfactory purity. In addition, the N,N-di(3,3-dimethylbutyl)-APM can not be recovered and used for the starting material, or etc., and therefore it is desired to suppress its production to the utmost. Since 3,3-dimethylbutylaldehyde is also problematic in odor (smell), it is desired to consume out the aldehyde as much as possible during the reaction. As one method of solving such an exhaust problem, a process for reacting APM with 3,3-dimethylbutylaldehyde in an excess of APM to the quantity of the aldehyde used might be considered. In such a case, where APM (unreacted) remains in the reaction solution (mixture), it would be desirable to establish a process for easily and efficiently separating APM from N-(3,3-dimethylbutyl)-APM, and thus separating and purifying each at a high purity. Unfortunately, such a process remains elusive.
Accordingly, it is an object of the present invention to provide a process for easily and efficiently separating the aspartame (APM) from N-(3,3-dimethylbutyl)-APM, and thus separating and purifying the two compounds each at a high purity, from an aqueous solution containing the APM and the N-(3,3-dimethylbutyl)-APM.
The above object and others are provided by a process for separating and purifying APM and N-[N-(3,3-dimethyl)-L-xcex1-aspartyl]-L-phenylalanine methyl ester, which entails subjecting an aqueous solution containing the APM and the N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine methyl ester to column chromatography using a non-polar, porous polymer-based resin to separate the APM from the N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine.
In order to solve the problem and achieve the object in the present invention, the present inventors have surprisingly discovered that APM can be easily and efficiently separated from N-(3,3-dimethylbutyl)-APM, and thus these two compounds can be each separated and purified at a high purity, from an aqueous solution containing the APM and the APM derivative, or the like by passing such aqueous solution or the like through column chromatography with the use of nonpolar highly porous polymer resin utilizing differences in affinity between them to nonpolar highly porous polymer resin used in the chromatography. Based on the above findings, the present invention has been completed. Thus, the present invention provides to a process for separating and purifying APM and N-[N-(3,3-dimethylbutyl)-L-(xcex1-aspartyl]-L-phenylalanine methyl ester (APM derivative), which entails: subjecting an aqueous solution containing said APM and said APM derivative, or the like to column chromatography with the use of a nonpolar highly porous polymer based resin to separate the APM from the APM derivative.
In the column chromatography operation, for example, an aqueous solution containing above-mentioned two types of compounds may be passed through the column filled with the above-mentioned resin effect separation of each other for purification of each compound, using known methodologies utilizing the differences between the above-mentioned two compounds in affinity to the resin, and specifically the property that APM is weak in affinity to the resin as compared to the APM derivative.
Now, a process of column chromatography, wherein the above-mentioned resin may be used in the form of mixture with another carrier or in combination with another carrier, is used by the present invention, however, the operation for the column chromatography must the above-mentioned resin filled in the column to effectively separate APM from the APM derivative in the mixture thereof.
In the present invention, the following aspects are also entailed:
1. The above process, wherein said nonpolar highly porous polymer based resin contains an aromatic polymer based resin or is an aromatic polymer based resin.
In this case, the above-mentioned aromatic polymer based resin used as the nonpolar highly porous polymer based resin may be employed for all the nonpolar highly porous polymer based resin contained in the column or for a part thereof, and these methods of using the resin are all involved in the present invention.
2. The above process, wherein said aromatic polymer based resin contains at least one resin selected from the group consisting of DIAION SP resin and DIAION HP resin.
3. The above process, wherein said DIAION SP resin contains at least one resin selected from the group consisting of DIAION SP 850 and DIAION SP 207.
4. The above process, wherein said DIAION HP resin contains DIAION HP 20 resin.
As used herein, xe2x80x9cDIAIONxe2x80x9d used for the carrier filled in the column is a trade name of Mitsubishi Chemical Corporation, and a carrier which may be essentially the same resin to any such DIAION in chemical constitution or composition and may have the same action to that of any such DIAION may be contained in the carrier having the above-mentioned trade name, even though it may not have such above trade name for the carrier.
5. In a process for production of N-(3,3-dimethylbutyl)-APM from APM as a starting material, the process for production of said APM derivative, i.e., N-(3,3-dimethylbutyl)-APM, wherein the APM or the fraction including APM separated and purified in any above process is used for said starting material of APM or a part thereof.
In this case, the process for the production as such is not particularly limited, and for its production, a process for production thereof in the reaction for reductive alkylation of APM with 3,3-dimethylbutylaldehyde is enumerated, for example.
6. An APM or an APM derivative obtained in the above process for the separation and purification in the present invention, or produced by being separated and purified using the above process.
7. A sweetener containing the above-mentioned APM derivative, or the above-mentioned APM derivative and a carrier usable for sweeteners, if required, and a process for imparting sweetness, comprising the step of: using the APM derivative for a material requesting sweetness, such as food.
The present invention is directed to a process for obtaining high purity APM and high purity N-(3,3-dimethylbutyl)-APM by separating and purifying each other utilizing differences between such two compounds in affinity to the nonpolar highly porous polymer based resin, in the separation of APM from N-(3,3-dimethylbutyl)-APM to purify each compound thereof from an aqueous solution (water based solution) containing the APM and the APM derivative, or the like, and particularly from the aqueous solution thereof.
The aqueous solution, or the like used for the starting material in the present invention may be in the form of the aqueous solution, or in the form of any solution which may be substantially the same solution thereto on an application to the column chromatography employed in the present invention. For the above-mentioned aqueous solution, for example, a water solution, a solution obtained from solvents which are dissolved or can be dissolved in water, such as methanol, ethanol, isopropyl alcohol, tetrahydrofuran and the like, which may include a solution obtained from a mixed solvent containing plural organic solvents which are dissolved or can be dissolved in water, and a solution obtained from a mixed solvent of water and such a solvent or such a mixed solvent selected from the organic solvents which are dissolved or can be dissolved in water are enumerated.
In the process for separating and purifying them in the present invention, it is desirable to operate the column chromatography according to the conventional or ordinal methods therefor. For example, a preferred operation is carried out by filling the nonpolar highly porous polymer resin in the column, and then passing through the column, preferably an aqueous solution containing APM and N-(3,3-dimethylbutyl)-APM to obtain a part of APM present as contained in the break through solution from the column, and further passing an appropriate eluting solvent (eluent) through the column to elute APM and N-(3,3-dimethylbutyl)-APM in this order.
For the aqueous solution containing APM and N-(3,3-dimethylbutyl)-APM, for example, a water solution obtained by substituting water for a solvent in the reaction solution obtained from the reductive alkylation of APM or a derivative of APM (for example, the derivative wherein the carboxyl group at the xcex2-position of the Asp residue in the APM is protected with a benzyl ester moiety) to produce N-(3,3-dimethylbutyl)-APM, a water solution which has dissolved the crude crystals of the product given from the reaction solution, the mother liquor therefrom or the like is cited. In addition thereto, any aqueous solutions which may contain both APM and N-(3,3-dimethylbutyl)-APM can be applied to the process in the present invention.
A concentration of APM or/and N-(3,3-dimethylbutyl)-APM in the aqueous solution is not particularly limited. An aqueous solution thereof in a concentration of about 0.1 to 1.5 g/dl is desirable for the separation and purification thereof. For the nonpolar highly porous polymer based resin used in the present invention, any resin which may be known as the nonpolar highly porous polymer based resin or the nonpolar highly porous polymer resin, or which may be essentially the same resin in structure or in function thereto may be employed. Among them, an aromatic polymer based resin is preferably employed, and for example, those made of the polymer of styrene and divinylbenzene, such as DIAION HP 20, HP 21, SP 206, SP 207, SP 825, SP 850 and the like, which are all trade names and produced in Mitsubishi Chemical Corporation, Japan, may be used. Any resin chemically equivalent thereto may be used.
As used herein, the aromatic polymer based resins preferably employed in the present invention DIAION HP 20, HP 21, SP 207, SP 825, and SP 850, may be better understood by reference to the following Table 1:
Regarding the quantity of the resin used, for example, in the case of DIAION SP 207 or DIAION SP 850, it is sufficient that the resin may be employed in a volume of about 0.1 times ({fraction (1/10)}) the volume of the aqueous solution in a concentration of N-(3,3-dimethylbutyl)-APM therein having about 1 g/dl. For the eluting solvent (eluent), water, alcohols such as methanol, ethanol, isopropyl alcohol, etc., a mixed solvent containing water and at least one alcohol at an any ratio optionally selected and the like are enumerated. For example, at the time of the operation for adsorption utilizing the aqueous solution containing APM and N-(3,3-dimethylbutyl)-APM, a part of APM present may be obtained in the break through solution from the column, and thereafter APM is obtained by the elution with water as the eluting solvent, and furthermore N-(3,3-dimethylbutyl)-APM is eluted from the column by the elution with alcohol(s) or a mixed solvent containing water and at least one alcohol as the eluting solvent. Accordingly, APM can be separated from N-(3,3-dimethylbutyl)-APM and also each compound can be separated and purified by taking properly fractional collections from the eluted solution.
The eluted solution containing either APM or N-(3,3-dimethylbutyl)-APM thus collected, may be recovered for each compound in the form of the solution and may be used directly for the each compound again, or from the solution, further the crystals for each compound at a high purity may be obtained by known purifying means such as concentration, crystallization and the like, where necessary, from the solution.
Now, a nonpolar highly porous polymer resin is lowered in adsorptive power when the operations for separation with the use of the resin are carried out about 30 times. In such case, an operation for regeneration of the resin used is needed. For regeneration, for example, the resin used and to be reproduced may be washed with a mixed solution consisting of the same quantity of two solutions obtained from 50% by volume of water alcohol solution and 1 mol/l sodium hydroxide (caustic soda) aqueous solution, i.e., a volume ratio of 1:1. In these regeneration methods, the resin can be used again and repeatedly.
There is no difficulty, when N-(3,3-dimethylbutyl)-APM separated and purified, or produced in the process of the present invention is used for a sweetener or food additive or in the production thereof. For example, known methods for production of sweeteners or food additives or for use thereof can be applied in such case using conventional amounts of APM or the APM derivative.